The effect of metoclopramide on intestinal muscle responses and the peristaltic reflex in vitro.

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The effect of metoclopramide on intestinal muscle responses and the peristaltic reflex in vitvo FRANCIS K. QKWUASABA~ AND JOHNT. HAMIL'TON~ Depnrtrnent of Pharn~acologp,U~tiversityo f Westerrt O~ttcrrio, London, Onr., Cn~~ucla N 6 A 5CI Received September 11. 1975 OKWUASABA. F., and HAMILTON, J. T. 1976. The effect of metoclopranlide on intestinal muscle responses and the peristaltic reflex in tlitro. Can. J . Physiol. Pharmacol. 54, 393404. Metoclopramide (Mcp) is known to facilitate gastrointestinal emptying irt vitw and to stimulate various isolated intestinal muscle preparations. On the guinea pig ileum, taenia coli, rabbit ileum and rat duodenum, Mcp increased the tone and responses to acetylcholine, carbachol and nicotine; hacl no effect on responses to histamine, potassium chloride and prostaglandin El; decreased responses to 5-hydroxytryptamine ( 5-HT). Atropine, methysergide, morphine, and tetrodotoxin, alone or in combination, partially blocked the stimulatory responses to Mcp, but hexamethonium. rnepyramine and indomethacin did not. Mcp ( 1.0 p M ) lowered the threshold for elicitation of the peristaltic reflex to a subthreshold intraluminal pressure (2.5 crn water), facilitated the peristaltic response to threshold pressures (3-4 cm water) and restored the reflex in fatigued preparations, but not that depressed by cooling to 24 "C. During block of peristalsis by atropine. hexamethonium or methysergide (applied serosally) 5-HT (0.25 piM) but not Mcp (1.0 p i l l ) effectively restored the peristaltic reflex, but neither antagonized the inhibition by morphine or procaine acting serosally. However, Mcp (1.0 p.M) re-established peristalsis inhibited by a high concentration of 5-HT ( 4 x 10 p M ) . These results d o not support the hypotheses that the sti~nulatory action of Mcp is entirely dependent on either peripheral sensitization of muscarinic receptors or an action on tryptaminergic mechanisms but are consistent with our previous conclusion that an additional component may be a blockade of some intrinsic inhibitory (possibly purinergic) substance normally restraining intestinal motility or tone.

Intr~ducti~n Studies have shown that Mcp (2-diethylaminoethyl - 2 - metIloxy - 4 - amino- 5 - chlorobenzamide) stimulates upper GIT nlotility in a synchronous onward fashion. While stimulating gastric pcristalsis (Grivaux et cil. 1964; Margieson ct (11. 1966) and small intestinal movement (Howarth et RI. 1967; James and Hume 1968 ; Kreel 1970) it relaxes the pyloric sphincter; hence its usefulness in radiological examinations. Probable mechanisms underlying the stimulaABBREVIATIONS: Mcp, metoclopramide (2-diethylaminoethyl-2-methoxy-4-amino-5-chlorobenzamide ) : 5-HT, 5-hydroxytryptamine crcatine sulfate; GIT, gastrointestinal tract; PGE,, prostaglandin El: T'TX, tetrodotoxin; A'TP, adenosine triphosphate; ADP, adenosine diphosphate; ECs,. mean effective concentration; HEXA, hexamethonium. 'Supported by Mid-West Nigerian Government Scholarship. 'Supported by Medical Research Council of Canada Term Grant to John T. Hamilton.

tory effect of Mcp on isolated GIT preparations have been proposed from studies on different species such as human intestinal longitudinal and colonic circular muscle strips (Eisner 1968), rabbit ileum and colon (Coulland and Leveque 1966), rat duodenum and jejunum (Hukuhara et 01. 1966), guinea pig ileum (Fontaine and Reuse 1973) and colon (Beani et RI. 1970). However, the depth of pharmacological analysis on these smooth-muscle preparations apFears limited. Effects involving peripheral cholinergic sensitization of the smooth muscle to acetylcholine and (or) on tryptaminergic nlechrtnisms (Hukuhara et (11. 1966; Bianchi et ul. 1970; Fontaine and Reuse 1973) have also been implicated, although central nervous system actions have been suggested (Justin-Besanpn and Eaville 1964; Chevrot et al. 1965). Since we have observed (Qkwuasaba and Hamilton 1975) that Mcp has a differential and selective antagonizing effect on the inhibitory


394

CAN. J. PHYSIOL. PHARMACOL.

action of purine nucIeotides on the GIT, it was decided to extend these studies using different agonists and antagonists on the same preparations (viz. guinea pig taenia coli, rabbit ileum, rat duodenum and Trendelenburg preparation of the guinea pig ileum) and include investigations of the effects of Mcp on peristalsis during exposure to specific antagonists, to cooling and to high concentrations of 5 - H T .

Methods and Materials Prepuratiolts Male guinea pigs wcighing 300-400 g; male New Zealand strain rabbits weighing 1-3 kg and male albino rats weighing 130-150 g were killed by a blow on the head, exsanguinated and the abdomen opened. Lengths (2-3 cm) of taenia coli from the guinea pig were dissected free from the circular muscle. Sections of the ileum from the guinea pig and rabbit, about 3 cm in length, were dissected free from thc mesenteric attachments, whilst sections of the duodenum about 2-3 cm in lcngth from the rat were removed with minimal amount of mesentery attachcd. These prcparations were mounted in a 10 ml organ bath containing Krebs' solution at 37 "C. gassed with 95% oxygen and 5% carbon dioxide. Thirty minutes wcre allowed for equilibration before exposure to drugs. Longitudinal contractions were recorded auxotonically via a Harvard Apparatus Muscle Transducer o n a Rikadenki (model B- 14 1) recorder. Peristalsis was studied by the interrupted method described by Trendelenburg (1917). This preparation was set up in a 65 ml organ bath containing Krebs' solution at 37 "C, gassed with 95% oxygen and 5 % carbon dioxide. The intraIumina1 pressure changes were monitored by a pressure transducer (Statham 4493-P23SC) connected after ampIification to one channel of the rectilinear Rikadenki recorder. Changes in Iength of the tissue (longitudinal contractions) were monitored as described above. The peristaltic reflex was initiated by increasing the intraluminal pressure to 3-4 cm of water above that of the atmosphere at 15min intervals for a period of 6 0 s.

VOL. 54, 1976

Statistical A~talysis In the studies with applied excitatory agonists, responses to each concentration of the agonist are expressed as percentages of maximum contraction obtainable in the absence of Mcp. Regression lines wcre calculated using the least-squares method (Wonnacott and Wonnacott 1972). Data points on the regression lines were computed using a Wang 520j600 calculator (programme number 21 33 ) . Log concentration (pi${)- percentage maximum response curves were constructed for each agonist using the pooled results from 7-10 preparations. The 95% confidence intervals and significance (determined by the Student's t test for paired data) between EC;,,s were calculated as described by Wonnacott and Wonnacott (1972). Also, mean ratios of the EC:,,s (potency ratios), their ranges and 95% confidence intervals were computed for each agonist in the presence of Mcp.

Results Effect of ThreslzoHd Concentrations of Mcp an tlzc Guinea Pig I i e ~ ~ m Tueniu , Caii, Rabbit Iiezmz and Rat Duodenurn Mcp produced a dose-related stimulation of the Magnus preparation of thc guinea pig ileaam in conceiltrations up to 80.0 pM, whereas higher concentrations produced smaller responses. No tachyphylaxis was observed with the lower doses provided that drug administration was followed by careful washing and by a suitable time interval (28-38 min). With concentrations between 1.5 and 30 pM Mep two qualitatively different types of responses could be identified: (a) an increase in muscle tone, reaching its maximum in a few seconds, which normally was maintained as a tonic contraction over the 1-min period of contact; or (b) a fast, short-lasting increase in tone which was followed by a period of spontaneous activity throughout the I-min observation time. The guinea pig isolated taenia coli muscle preparation responded reproducibly to 2.5-30 &laterials Freshly prepared solutions of the drugs were dis- p M Mcp with a sustained increase in tone and solved in I&ebs9 solution of the following con~position phasic activity. The qualitative response varied ( m M ) : KC1 4.75, CaCl: 2.54, MgSO, 1.19, KH-PO, with the drug concentration, although responses 1.19, NaCl 118, N a H C 0 3 25, and glucose 11. Drug were reproducible at all dose levels tested, proconcentrations are expressed as final bath concentrations. The agents used were: acetylcholine iodide vided that 20-30 min was allowed between addi(B.D.H.), atropine sulphate QB.D.HI), carbaminsyl- tions of the drug. In a large percentage of expericholine (carbachol chloride) (B.D.H.), hexame- ments 5.0-38 pM Mcp gave rise to a bisphasic thonium bromide (Sigma), histamine acid phosphate response consisting of a rapid, short-lasting (B.D.H.), 5-HT (B.D.H.), indomethacin (Merck, inhibition of tone, followed by a sustained conmaIeate (Psulenc) Sharp and Dohme), mepyramine methysergide bimaleatc (Sandoz), morphine sulphate traction. The magnitude of the inhibitory phase (B.D.H.), nicotine hydrogen tartrate (B.D.H.), potas- appeared to be directly related to the tone, but sium chloride (Mc.4rthur Chemical), prostaglandin El the stinlulatory phase was not proportional to the (Upjohn), tetrodotoxin (Sigma) and metoclopramide initial relaxation. monohydrochloride (Lot No. W.S. 111 - 130 HH620, On thc rabbit ileum 3-25 p M Mcp elicited consupplied by Delmar Chemicals Ltd., Montreal, to the centration-related contractions with normal late Dr. J. M. Barker).

OKWUASABA AND HAMILTON


TABLE1. ECjo values for efFects of Mcp on intestinal smooth muscle preparations ECjo* X 10-\ M (957; confidence limits)

Smooth muscle preparation

--

Guinea pig ileum (tt Taenia coli 01 -(pz Rabbit ileum 01 Rat duodenum

121)f 121) 87) 87)

7 - 5 (7.29-7.65) 9.5 (9.39-9.67) 8.2 (8.13-8.38) 8.4 (8.23-8.53)

*ECjo values calc~~latcd f r o n ~vaiues from 10-1 2 preparationr.

t r r , number

of data points.

rhythmic activity superimposed. On the rat duodenunl 5-30 pM Mcp caused a sustained increase in tone; again, the respollses were concentration-related and reproducible. The EGsos for the stiinulatory effects of Mcp on the four preparations are given in Table 1. Eflect of Mcp otz Respo~zsesto ExcitutorjAgonists The results for all four preparations are summarized in Table 2. Subthreshold conccntrations (0.1 and I .O pM) of Mcp produced significant increases (p < 0.001) in the acetylcholine-, carbachol-, and nicotine-induced contractions in each of the four preparations, but did not alter the maxirnunl responses. Mcp significantly reduced the contractions evoked by 5-HT but had no effect on the dose-response curves for histamine, potassium chloride, or PGE1. The dose-response curves with these agonists on the guinea pig taenia coli preparation are illustrated in Fig. 1. Although the curves were parallel over

CONCENTRATION @M(log scale)

FIG.1. The effect of subthreshold concentrations of Mcp on the concentration-response curves of spasmogens on the guinea pig taenia coli: ( a ) acetylcholine, ( 6 ) carbachol. ( c ) nicotine. (ti) histamine, ( e ) 5hydroxytryptamine. ( f ) potassium chloride. and (g) prostaglandin El. Curves:. control. - ( a ) ; 0.1 pM Mcp. - - - ( b ) ; 1.0 p M Mcp. - - -. The tissue was incubated in h4cp for 5 min. The E;C;*, values from regression lines were obtained by pooling the results from 7-10 preparations. Horizontal lines on EC:,,, represent the range calculated as described in the text.

TABLE 2. ECjo values for effect of subthreshold concentrations of Mcp on response of smooth muscle preparations to various stirnulatory agonists (A) guinea pig ileum, (13) guinea pig taenia coIi. (C) rabbit ileum, (D) rat duodenunl -

- -

ECj8 X 1QP8M (95% confidence limits) Muscle Agonist preparationa

hlcp present ( J M ) I I(

Agonist ( X )

A Acetylcholine

I01

Carbachol

9 '7

Nicotine

9'7

Histamine

Y0

5-Hydroxytryptamine

97

Potassium chloride

75

Prostaglandin El (PGE,)

75

2.2 (2.11-2.34) 31.6 (20.90-33.21) 151.4 (142.17-160.33) 2.1 (1.92-2.22) 1.8 (1.61-1 .X9) 3.5 X 105 (3.39-3.67) 3.9 (3.71-3.99)

0.1 ( Y )

1 .O

Mean ratio of ECso (X/Y) (range)"

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CAN. J. PHYSIOL. PHARMACOL. VOL. 54, 1976

TABLE 2. ( C ' O I I C I I I ~ C ~ ~ )


EG;,oh x 10 ""nl(95';

1confidence limits) Mcp present (cLM)

Muscle Agonist preparationn

Agonist (X)

tzc

0.1 (Y)

1 .O

Mean ratio of EC50 ( X / Y ) (range)d

B Acetylcholine

Carbachol Nicotine His tarnine 5-Hydrsxytryptarnine Potassiunl chloride IZrostaglandin E, (I'GE,) C Acetylcholine

Carbachol Nicotine Histamine 5-Hydroxytryptarnine Potassiunl chloride Prostaglandin El (PGE,) D Acetylcholine

Carbachol Nicotine Histamine 5-Hydroxytryptamine Potassium chloride Prostaglandin El (PGE,) -

-

-

-

- --

-

-

--

-

-

-

C(A) Guinea pig; (B) guinea pig taenia coli; (Cj rabbit ilcum; (D) rat duodenum.

%C:o values from regression lines obtained by pooling results from 7-10 preparations. CNutnber of data poir~ts. d ~ n n calculated ~ e as (ECso(X) - 95 confidence interval) !(ECso(Y) 95 % confidcrlce interval) to (EC;a(X) -$- 95 (ECao(Y ) - 95 % confidence interval). NOTE:Significantly diflerent from (X)a t P < 0.001.

+

the range 20-SO%, it was noted that 5-HT failed to elicit a ~naxirnalresponse in the presence of Mcp, particularly at the higher concentration.

confidence intervali!

Efects of Arlttrgonists on Responses to Mcl, rrnd E..ccitatory Agonists Qualitatively similar results were obtained on

OKWUASABA A N D HAMILTON


(b)

CONCSNTRATCU

@

COHCEUTRAIION

,&

CONCENTRATION

pU

The effect of antagonists alone and in combination on the response to different concentrations ( p M ) of Mcp on the guinea pig taenia coli. In a-e. open bars represent control response to Mcp. ( n ) Hatched bar, atropine (0.1p M ) ; dotted bar, atropine (0.1pk?) and 7TX (0.1 p M ). ( b ) Hatched bar, rnethysergide ( 1.0 p M ) ; dotted bar, rnethysergide ( 1.0 p M ) and atropine (0.1pM). (c) Hatched bar. TTX (0.1p M ) ;dotted bar, TTX (0.1p M ) and atropine (0.1 pM). ( d ) Hatched bar, morphine (50p M ) ; dotted bar, morphine (50p M ) and methysergide ( 1.Q p M ) . ( e ) Hatched bar, hexamethoniurn (50 p M ) . ( f ) Hatched bar, rnepyramine ( 1.0 p M ) . ( g ) Hatched bar, indomethacin (80p M ). FIG. 2.


398


all four preparations and those on the taenia coli are presented as bar graphs in Fig. 2. Hexamethonium (58.0 pM), mepyrarnine (1.0 pM) and indoinethacin (84.0 plW) had no significant effect on the responses tc) Mcp on any of the preparations. This comcentratiora (50 pM) sf hexamethomium, however, did block the csntractions evoked by 8.6-1.0 pM nicotine on the guinea pig ileum, and taenia coli, rabbit ileum, and rat duodenum. Mepyrarnine (1.0 yM) also abolished the responses to histamine (0.01 and 8.08 pM) on the guinea pig ileum, (0.1 and 0.06 plW) on the taenia coli, (0.01 and 0.08 pM) on the rabbit ileum and (0.03 and 0.20 pM) on the rat duodenum. Indomethaciaa (94.8 pM) reduced by 88-95% the responses to PGEl (0.004 and 0.01 pM) on the guinea pig ileum, (0.002 and 8.008 pM) on the taenia coli and (0.006 and 0.01 pM) on the rabbit ileum. Atropine (0.1 yM) partially and significantly inhibited the stimulatsry action of Mcp by 70-75%, on all four smooth n~usclepreparations, although it was unable to exert a csnaplete inhibition. However, this concentratiola of atropine completely abolished the contractile responses to acetylcholine (0.01-0.60 pM) on all the intestinal preparations. Similarly, methysergide produced a partial blockade (15-2574,) of responses to Mcp on all four preparations at a concentration (1.0 pM) which reduced but did not abolish the response to 5-HT (0.01-0.60 pM). To determine whether the atropine- and methysergide-resistant components of the Mcpinduced contractions involved neuronal elements, the interactions between Mcp and TTX (0.1 pM) and Mcp and morphine (50.0 pM) were investigated. TTX produced a partial (70-757,) but significant (p < 0.001) blockade of responses to Mcp in all the preparations. Similar results were obtained witla morphine, the degree of inhibition being 60-70%. In another series of experiments, the eEects of two antagonists administered together were studied on Mcp-induced contractions. The results obtained showed that the simultaneous addition of two antagonists (atropine methysergide; atropine TTX; atropine morphine; methysergide TTX; or methysergide morphine) although resulting in a further and significant (4B < 0.001) reduction in the responses to Mcp, fttiled in all instances to abolish the responses.

+ +

+

+

+

AFTER

F,

FIG.3. Effect of Rlcp on the response of TrendeIenburg preparation to threshold (3.0 cm water pressure) and subthreshold pressures ( 1.0 and 2.5 cm water pressure). 1P, intralurninal pressure changes; L, amplitude and frequency of the longitudinal muscle contraction proper; G, graded contraction of the longitudinal muscIe. Upper trace: before addition of Mcp into the bath. Lower trace: after incubation of the tissue with Mcp for 5 min. The intraluminal pressure was elevated for a period of 1 min.

Mcp and Peristaltic Activity (Trendelenburg Preparntisrz) A typical response of the ileurn under subthreshold pressures sf 1.0 and 2.5 and the threshold pressure of 3.0 crn of water, before and after pretreatment with Mcp is shown in Fig. 3. At 1.8cm H 2 8 , in the absence of Mcp,

OKWUASABA AND HAhlILTBN


(a)

1

1-

? 7 I

---

FIG.4. ( a ) Effect of Mcp on peristaltic activity of Trendelenburg preparation during fatigue. before addition of Mcp into the bath (C) and after Mcp (1.8 p M ) , observed during elevation s f intraluminal pressure (3-4 cm water pressure) for 120 and 188 s, respectively. ( b ) Effect of Mcp ( 1.0 p M ) and 5-HT (0.25 p M ) on peristaltic activity of Trendelenburg preparation inhibited by cooling the preparation to 24 " C . Control response ( C ) at 36.5 "C. The intraluminal pressure was elevated for a period of 1 min. (c.1 Effect of Mcp ( 1.0 p114) on peristaltic activity of Trendelenburg preparation inhibited by 5-HT (4 x 10 p M ) . Control response = C. The intraluminal pressure (3-4 cm water pressure) was elevated for a period of 1 min.

only graded reflex contraction of the longitudinal muscle was observed, whereas at 2.5 cin H 2 0 , fast contractions of loi~gitudinal muscle were superimposed on the graded reflex contraction without eliciting a co-ordinated peristaltic reflex proper. The administration of 1.0pM Mcp, however, consistently evoked peristaltic activity with a stimulus of 2.5 cm water pressure (six experiments) and with a stimulus of 3-4 cm H2Q,

Mcp enhanced the amplitude and frequency of the longitudinal muscle contraction proper and the frequency of the intraluininal waves (10 experiments). Although not shown in this figure, Mcp more often than not also increased the amplitude of the graded contraction. In a series of five experiments, the effect of Mcp was investigated on 'fatigued' preparations, fatigue being defined as that state in which


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CAN. J . PIIYSIOL.

PHARMACOL. VOL.

54, 1976

FIG.5. Effect of Mcp (1.0 p M ) (left) and 5 PIT (0.25 p M ) (right) on peristaltic activity of Trendelenburg preparation inhibited by ( t z ) atropine (0.1 p M ) , ( h ) HEXA (5.4 p M ) , ( c ) methysergide (10 pM). (rl) morphine ( 5 0 p M ) , ( e ) procaine (4.0 p M ) . Control response = C. Intraluminal pressure (3-4 cm water pressure) was elevated for a period of 1 min.

peristaltic activity ceased following 2 min of increased intraluillinal pressure. Mcp (1.0 pM) antagonized the effect of fatigue by allowing the return of normal peristalsis which lasted for the entire 3-rnin observation period (Fig. 4a). It is known that cooling the intestinal preparation reduces the amplitude of the graded contraction and abolishes the emptying phase of the peristaltic reflex in the guinea pig ileum (Kosterlitz and Robinson 1957). Similar responses to lowering the bath temperature to 24 "C are illustrated in Fig. 4h. The addition of Mcp (1.0 pM) to the bath did not cause the reappearance of peristalsis: however, whcn a dose of 0.25 pM 5-HT was added to the bath, there was a partial return of peristaltic activity, an effect of 5-HT similar to that reported by Biilbring and Crema (1958). Figure 4c shows that pretreatment with a high dose of 5-HT (40 pM) resulted in transient stimulation of peristaltic activity followed by blockade and that pretreatment with Mcp (l.OpM) partially antagonized the inhibitory effect of 5-HT.

EfJ'ict of Mcp and 5-HT on Peristulsis Inhibited by Antugonisis Atropine (0.1 p M ) , hexarnethoniunl (5.4 pM), methysergide (10.0 pM), morphine (50 pM), and procaine (4.0 &M) reduced but did not abolish the graded contraction of the longitudinal muscle, whilst thc peristaltic reflex proper was abolished by each agent. A typical response t o cach antagonist in the absence and presence of Mcp (1.0 pM) is shown in Fig. 5u-c. It can be seen that Mcp added to the bath 10 min before the administration of any of the antagonists failed to restore the peristaltic activity. Similar experiments were conducted after pretreatment with 5-HT (0.25 pM). Figure 5cr-c. shows that 5-WT restores partially peristaltic activity inhibited by atropine, hexarnethoniurn and methysergide but was ineft'ective on peristaltic activity which has been abolished either by morphine or procaine. Discussion The results obtained from this investigation are consistent with earlier reports that Mcp



possesses a stirnulatory action on the GIT in vivo in man and animals and further confirms the observations of Beani et ak. (1970) and Fontaine and Reuse (1973) on the guinea pig colon and ileum, respectively. The observation that Mcp at subthreshold concentrations of 0.1 and 1.O plM significantly potentiated the responses to acetylcholine, carbachol and nicotine on the guinea pig ileu~m, taenia coli, rabbit ileum and rat duodenum suggest that the drug somehow enhances the cholinergic influence on the intestinal smooth muscle. This observation is in agreement with those reported by Eisner (1968) on human CIT, Beani et ak. (1978) on the colon and Fontaine and Reuse (1973) on the ileum of the guinea pig. The enhancement of cholinergic influence may be due to: (i) acetylcholine release, by an action on preganglionic fibres and (or) on the postganglionic terminals; (id) sensitization of n~uscarinic receptors to acetylcholine; (iii) suppression of mechanisms controlling the amount of available acetylcholine. Against the first hypothesis is the finding of Beani et nl. (1970) that Mcp did not change acetylcholine (3 X 18-"/ml) release at least from the colon at rest or during pelvic nerve stimulation. Objection to the third postulate arises from the c9bservation by the same authors that Mcp (3 X 18-0 g/ml) did not affect the total cholinesterase activity in the guinea pig colon. The observation, that Mcp, at concentrations which potentiated the responses to cholinergic agonists, did not alter significantly histamineinduced contractions on all four intestinal muscle preparations, ruled out the possible involvement of histamine receptors and of a nonspecific sensitization in the genesis of Mcp contractions. In contrast, however, Birtley and Baine (1973) have reported sensitization by Mcp of guinea pig ileal longitudinal and circular muscle strips to acetylcholine and histamine to a similar degree. The present study shows that the stimulatory responses t o Mcp were partiaIly antagonized by atropine (8.1 pM), a result at variance with those of Hukuhara et ul. (1966) who reported that on the rat and rabbit duodenum Mcp-stimulating responses were insensitive to atropine. Our result with atropine suggests that part of the action of Mcp involves a specific muscarinic receptor and is not simply a nonspecific effect as suggested by Bjrtley and Baine (1973) since

40 1

responses to histamine, potassium chloride and PCEl were not significantly altered on all four preparations studied. Sensitization to acetylcholine does not appear, hc)wever, to explain all the action of Mcp because part of it was found to be atropineresistant. Responses to added 5-PIT were partially antagonized on all the preparations and a ~maxi~mum response to 5-HT was never obtained in the presence of Mcp. Fontaine and Reuse (1973) suggested that Mcp releases 5-WT and is unable to potentiate added 5-HT on the guinea pig ileum due to tachyphylaxis resulting from the liberated 5-HT. Against the hypothesis of 5-PIT release by Mcp is the observation that the respcmses to 5-HT on all the preparations were not potentiated but antagonized by Mcp. If Mcp were releasing 5-HT from storage sites, an enhancement of the response to exogenous 5-HT wculd have been expected. In addition, 5-HT is known to stimulate both muscular receptors (D-receptors) and neural receptors (M-receptors) in most intestinal preparations (Caddum and Picarelli 1957), the effect on M-receptors usually resulting in acetylcholine release. The lack of any observable increase in tone of the preparations when Mcp was added to the bath 5 min before the addition of 5-HT in our experiments and the reports by Beani et al. (1970), that there was no increase in acetylcholine release following exposure to Mcp on the colon, do not lend support to the hypothesis that Mcp releases 5-HT. A question which remains to be answered, however, is how susceptible are the stores of 5-HT to the releasing action, if any, of Mcp because it is well known that intestinal 5-WT stores show a relative resistance to the depleting effect of reserpine (Carlsson 1966 Bennett et al. 1966). As an alternative hypothesis it is proposed that Ncp may act as a partial agonist on the same receptors as 5-HT. The observation that higher concentrations of Mcp (1 .O plM) had little further antagonistic action on responses to 5-HT suggest a ceiling effect indicative of receptor saturation with a compound of low intrinsic activity or efficacy and is consistent with the lack of ability of 5-HT to elicit a maximal response in the presence of Mcp. Additional support for this idea stems from the observation that Mcpinduced contractions were partially antagonized by rnethysergide, a specific and competitive antagonist of 5-HT on D-receptors, and mor-


402


phine which has been reported to inactivate the M-receptors. Although methysergide was more effective in blocking 5-HT responses than those elicited by Mcp, the observed partial inhibition of Mcp responses by both types of 5-HT antagonists demonstrates that Mcp is capable of interacting with a tryptaminergic mechanism. It was expected that combinations of antagonists would completely block the response to Mcp if this were due entirely to interaction with tryptaminergic as well as cholinergic receptors. Combinations of antagonists, however, completely blocked responses to 5-HT without effecting a total blockade of Mcp. This resistant component of Mcp is not due to PGE synthesis or release since we have also shown that Mcp did not alter the response to PGEr on any of the intestinal preparations employed. Vane (1971) demonstrated that low doses of indomethacin specifically inhibit PGE synthesis (EC5, = 0.27 pgjml), while Sorrentino c.t crl. (1972) reported complete inhibition with 1.0 pg/n~l.Indomethacin (2.8 pM) reduced but did not completely abolish responses to PCE1, whereas the response to acetylcholine, histamine and 5-HT were unaffected. Similarly, the dose-response curve to Mcp was unaffected even with higher concentrations (84 pM) of indomcthacin. Mcp contractions are not dependent on preganglionic cholinergic neurones or nicotinic receptors on the ganglion cells as demonstrated by the lack of effect of hexamethonium. Similar observations havc been reported by various authors on other intestinal preparations. Our result that Mcp did not change potassium chloride responses indicates that no coinponent of its action is mediated through depolarization of smooth muscle cells. Mcp (1 .O pM) partially antagonized the inhibitory effect of a high concentration of 5-HT on the peristaltic reflex. At high concentrations 5-HT has been shown to produce inhibition of several GIT preparations, including large bowel, stomach and ileum and it has recently been proposed (Biilbring and Gershon 1967; Drakontides and Gershon 1968 Gershon 1970) that 5-HT acts as the transmitter to noiladrenergic inhibitory neurons. ATP or ADP has recently been suggested, on strong evidence, to be the putative transmitter in such inhibitory neurones (Burnstock et ul. 1970; Axelsson and Holmberg 1969). It is possible, therefore, that at post-

synaptic sites Mcp antagonizes the inhibitory transmitter released by 5-HT. The residual efl'ect of Mcp in the prescnce of combincd antagonists could be accounted for if there was a resting relcase or leakage of inhibitory transmitter to modulate the tone of smooth muscle preparations. Our systematic study (Okwuasaba and Hamilton 1975) showing that Mcp had a selective blocking effect on the inhibitory responses to the purinergic compounds ATP, ADP, and adenosine strongly supports such a hypothesis. In the presence of Mcp peristalsis could be elicited by an otherwise subthreshold pressure, and at threshold pressures (3-4 cm HzO), as well as in fatigued preparations, the peristaltic activity was facilitated. According to Chujyo (1963) and Katie and VaragiC (1968) stretching or raising the intraluminal pressure leads to the release of acetylcholine. It may be that Mcp sensitizes the smooth muscle to the released acetylcholine which results in increased tone in the longitudinal muscle and elevated intraintestinal pressures sufficient to trigger peristaltic activity. Mcp was without effect on preparations in which peristaltic activity had been abolished by cooling or exposure to atropine, hexamethonium or methyscrgide; 5-HT (0.25 pM) did, however, par tially antagonize the effects of these agents. The inability of Mcp or 5-HT to restore peristalsis after it had beenblocked by morphine or procaine demonstrates that neural conduction is a requirement for the facilitatory effects of Mcp and 5-HT. The results with 5-HT support the theory that it acts by facilitating the transmission of impulses from aflerent to efferent parts of the reflex arc (Trendelenburg 1956; Beleslin and Vara~iC1957; Biilbring and Crema 1958). That Mcp did not restore the reflex is consistent with the fact that its effect on smooth muscle strips was not altered by the ganglion blocking agent, hexamethonium, and further suggests that Mcp does not facilitate cholinergic transn~ission at ganglia. In addition, the results with atropine and methysergide further indicate an intcraction between Mcp and mechanisms sensitive to these agents and are consistent with Mcp being a partial agonist for 5-HT receptors. Moreover, as a partial agonist Mcp would not be expected to overcome the inhibition resulting from a potent antagonist such as methysergide.



It is concluded that the action of Mcp is indeed a complex one, the observed response being determined by the ability of Mcp to scnsitize muscarinic sites, interact in some way with tryptaminergic mechanisms and to block intrinsic inhibitory, possibly purinergic, transmission at post-synaptic sites (Okwuasaba and Hamilton 1975). Acknowledgments We are indebted to the late Dr. J. M. Parker for his suggestion that metoclopramide was an agent worthy of further basic investigation, the technical assistance of Mr. C. %. Paterson, and the photography by Mr. J. Klaase. We also wish to thank Dr. C. W. Gowdey for his kindness in reviewing this manuscript. AXELSSON, J., and HOLMBERG, B. 1969. The effects of extracelIularly applied ATP and related compounds on electrical and mechanical activity of the smooth muscle taenia coli from guinea pig. Acta Physiol. Scand. 75, 149-156. BEANI,L., BIANCHI,C., and CREMA,C. 1970. Effects of metoclopramide on isoIated guinea pig colon. Peripheral sensitization to acetylcholine. Eur. J. Pharmacol. 12, 320-3 3 1. BIANCHI, C., BEANI,L., and CREMA, C. 1970. Effects of metoclopramide on isolated guinea pig colon. Interference with ganglionic stimulant dr~igs.Eur. J. Pharmacol. 12, 332-341. BELESLIN,D., and VARAGIC, V. 1958. The effect of cooling and of 5-hydroxytryptamine on peristaltic reflex of the isolated guinea pig ileum. Br. I. Pharmacol. 13,266-270. BENNETT, A., BUCKNELL, A,, and DEAN,A. C. B. 1966. The release of 5-hydroxytryptamine from the rat stomach in vitro. J. Physiol. (London), 182, 57LC

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BIRTLEY, R. D. N., and BAINE,M. W. 1973. The effect of metoelopramide on some isolated intestinal preparations. Postgrad. Med. J. 49, 13-18. B~~LBRING, E., and CREMA, A. 1958. Observations concerning the action of 5-hydroxytryptamine on the peristaItlc reflex. Br. J . Pharmacol. 13, 444-457. BULBRING, E., and GERSHON, M. D. 1967. 5-hydroxytryptamine participation in the vagal inhibitory innervation of the stomach. J. Physiol. (London), 192,823-846. BURNSTOCK? G., CAMPBELL, G., SATCHELL, D., and SMYTHE,A. 1970. Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut. Br. J. Pharmacol. 40,668-688. CARLSSON, A. 1966. Drugs which block the storage of 5-hydroxytryptamine and related amines. I n Handbook of experimental pharmacology, 5hydroxytryptamine and related indolealkylamines. Springer-Verlag, Berlin. p. 529.

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CHEVROT, L., ROXY,G., and MENARD, J. C. 1965. Metoclopramide et routine radiologique digestive. Sem. Hop. Paris, 41, 1803-1805. CHU.JYO, N. 1963. Site of acetylcholine production in the wall of the intestine. Am. J. Physiol. 174, 196198. COULLAND, D., and LEVEQUE, J. 1966. Action du mktoclopramide sur differentes portions de l'intestin is016 de lapin et sur l'intestin in situ. Pathol. Biol. 14,963-965. DRAKONTIDES, A. B., and GERSHON, M. D. 1968. 5Hydroxytryptamine receptors in the mouse duodenum. Br. J. Pharmacol. 33,480-492. EISNER. M. 1968. Gastrointestinal effects of metocloprarnide in Inan. In viao experiments with human smooth muscle preparations. Rr. Med. J. 4, 679-680. FONTAINE, J., and REUSE,J. J. 1973. Pharmacologicd analysis of the effects of metoclopramide on the guinea pig ileum in vitro. Arch. Int. Pharmacodyn. Ther. 204,293-305. GADDUM, J. H., and PICARELLI, Z. P. 1957. TWOkinds of tryptamine receptors. Br. J. Pharmacol. 12, 323-328. GERSHON, M. D. 1970. The localization of S-hydroxytryptamine and its participation in neural inhibition of gastrointestinal smooth muscle. Jpn. J. Smooth Muscle Kes. 6, 113-1 15. GKIVAUX, M., CORNET, A., and WATTEZ, E. 1964. Metoclopramide in radiology of the digestive system. J. Physiol. (Paris) 26, 107-1 18. HOWARTII, F. H., COCKEL,R., and HAWKINS, C. F. 1967. Effect of metoclopramide upon gastric motility and its value in barium meal progress. Gut, 8, 635-636. T., NAKAYAMA, S., FUKUDA, H., and NEYA, HUKUHARA. T. 1966. Mechanism of action of ~netoclopramide on intestinal movements. Jpn. J. Smooth Muscle Kes. 2, 22-26. JAMES,W. B., and HUME,R. 1968. Action of metoclopramide on gastric emptying and small bowel transit time. Gut, 9, 203-205. JUSTIN-BESAN

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The effect of metoclopramide on mefloquine pharmacokinetics.

Enteric GABA: mode of action and role in the regulation of the peristaltic reflex.

The effect of metoclopramide and haloperidol on tardive dyskinesia [proceedings].

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The peristaltic reflex: an analysis of the nerve pathways and their pharmacology.

Stimulation of the peristaltic reflex by sodium chloride in guinea-pig isolated ileum.

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The effect of ethoxyquin on the intestinal transport of glucose and water in vitro.

The effect of Marcaine on muscle and non-muscle cells in vitro.

The effect of transureteroureterostomy upon conduction of peristaltic waves.

The effect of metoclopramide on neonatal gastric emptying.

Reflex responses of fusimotoneurons to sinusoidal muscle stretching.

The effect of crossed reflex responses on dynamic stability during locomotion.

On the measurement of vascular and respiratory smooth muscle responses in vitro.

Infant colic: the effect of serotonin and melatonin circadian rhythms on the intestinal smooth muscle.

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The effect of vasoactive intestinal peptide (VIP) on the contractile activity of human uterine smooth muscle.

Effect of insulin on human intestinal smooth muscle.

Reflex responses of gamma motoneurones to vibration of the muscle they innervate.

The effect of food and metoclopramide on the pharmacokinetics and side effects of bromocriptine.

Proceedings: The effect of chlorpromazine on carbachol binding to muscarinic receptors in intestinal smooth muscle.

Effect of dimeticone and pepsin on the bioavailability of metoclopramide in healthy volunteers.

The effect of bovine lactoferrin on muscle growth in vivo and in vitro.